41 Bull Vet Inst Pulawy 50, 41-45, 2006
ANTIBIOTIC RESISTANCE OF STAPHYLOCOCCUS AUREUS AND COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATED FROM BOVINE MASTITIS HULYA TURUTOGLU, SENAY ERCELIK AND DILEK OZTURK Department of Microbiology, Faculty of Veterinary Medicine, Akdeniz University, 15100 Burdur, Turkey e-mail:
[email protected] Received for publication September 22, 2005.
Abstract The antibiotic susceptibility test was carried out on 103 Staphylococcus aureus and 136 coagulase-negative staphylococci (CNS) strains. Only 35 of the isolates were susceptible to all antibiotics tested, while the remaining 204 isolates were resistant at least to one of the antibiotics. Among staphylococci, 18 isolates of S. aureus and 31 isolates of CNS were found phenotypically resistant to methicillin and these isolates were also resistant to penicillin G, ampicillin, amoxycillin and cloxacillin. Out of 68 S. aureus strains 55.9% were β-lactamase producers. β-lactamase producer isolates were 21.1% resistant to methicillin, but were 100% susceptible to amoxycillin/clavulanic acid and 97.4% susceptible to ampicillin/sulbactam. The differences observed in the efficacy of the antibiotics tested against staphylococci show that the antibiotic susceptibility tests should be performed together with the identification of the bacterial agents.
Key words: cows, mastitis, Staphylococcus aureus, antibiotic resistance. Staphylococcus aureus has been the main subject of studies on antibiotic resistance because of its importance for all forms of mastitis in dairy cows (4, 11, 14). However, in recent years, the incidence of reported coagulase-negative staphylococci (CNS) mastitis has increased substantially (3, 7, 18). In human medicine, antimicrobial multi-resistance is frequently encountered and methicillin-resistant S. aureus (MRSA) (2, 15) and methicillin-resistant CNS (MR-CNS) (2, 17) strains are among the most threatening bacteria involved in nosocomial infections. In veterinary medicine, however, MRSA as well as multi-resistant S. aureus strains are reported occasionally (13, 19). Penicillinase or βlactamase resistant penicillins such as methicillin and oxacillin are not used in veterinary medicine in Turkey except for cloxacillin that is used in products for intramammary administration in cattle. Therefore, testing the susceptibility to these antibiotics is not a routine procedure and infections with methicillin-resistant staphylococci (MRS) in animals may go undetected. The objective of this study was to determine the in vitro resistance rates of
staphylococci isolated from bovine mastitis to methicillin and other antibiotics commonly used in veterinary medicine.
Material and Methods Sample collection and microbiological analysis. The antibiotic susceptibility test was carried out on 239 staphylococcal strains (103 S. aureus and 136 CNS) isolated from milk samples which were collected from cases of mastitis in cow herds in Burdur province of Turkey during the years 2002-2004. Before sampling the teat ends were cleaned with alcohol swabs and allowed to dry. The first few streams were discarded and then 5 ml of secretion was collected in sterile tubes. Samples were cooled and immediately transported to the laboratory. From each milk sample, 0.1 ml was plated on Columbia blood agar medium (Oxoid Ltd, Hampshire, UK), containing 5% of sheep blood and 0.1% of esculin, and incubated at 37°C for 48 h. The isolates were identified by conventional methods, including Gram staining, colony morphology, haemolysis, tests for catalase, clumping factor, coagulase, DNAse, acetoin and anaerobic fermentation of mannitol. All the tests were performed as described by Koneman et al. (12). All the isolates were stored at -20ºC in trypticase soy broth containing 10% of glycerol. Prior to the testing, the isolates were twice serially cultured on Columbia blood agar medium, containing 5% of sheep blood, for 24 h at 37ºC under aerobic conditions. Antibiotic susceptibility test. Antibiotic susceptibility test was performed using disk diffusion method on Mueller-Hinton Agar (Oxoid) according to the National Committee of Clinical Laboratory Standards (NCCLS, 16). Ten colonies from the Columbia blood agar medium, incubated at 37ºC for 18 h, were suspended in 2 ml of sterile saline to a density approximately equal to McFarland Opacity Standard No. 0.5. A dry cotton wool swab was placed in the suspension and excess liquid was expressed against the inside of the tube. The bacterial suspension was inoculated onto Mueller-Hinton agar with the swab in such a way that the whole surface of the agar was covered. The
42 antibiotic disks, containing the following antibiotics: penicillin G (Oxoid, 10 iu), ampicillin (Oxoid, 10 µg), amoxycillin (Oxoid, 25 µg), ampicillin/sulbactam (Oxoid, 20 µg), amoxycillin/clavulanic acid (Oxoid, 30 µg), methicillin (Oxoid, 5 µg), cloxacillin (Oxoid, 5 µg), cefuroxime (Oxoid, 30 µg), neomycin (Oxoid, 30 µg), lincomycin (Oxoid, 15 µg), enrofloxacin (Oxoid, 5 µg), danofloxacin (Mast Diagnostics, Mast Group Ltd., Merseyside, U.K., 5 µg), gentamicin (Oxoid, 10 µg), trimethoprim/sulphamethoxazole (Oxoid, 25 µg), oxytetracycline (Oxoid, 30 µg), were dispensed on the surface of the medium and incubated aerobically at 37ºC for 18 h. The media, in which susceptibility to methicillin and cloxacillin was tested, were incubated at 35ºC for 24 h. The results were recorded as resistant or susceptible by the measurement of the inhibition zone diameter according to the interpretive standards of NCCLS (16). The reference strain used for antibiotic susceptibility assays was S. aureus ATCC 25923. β-lactamase testing. Only 68 of S. aureus isolates were tested for β-lactamase production using nitrocefin sticks (Oxoid, BR66A). The colonies in the inhibition zone of the penicillin disk on the Mueller-Hinton agar were touched gently with the sticks and the sticks were incubated at room temperature and under humid condition for 1 h. Development of a dark pink-red colour were considered to indicate a positive response (20).
Results The antibiotic resistance rates of S. aureus and CNS isolated from bovine mastitis are detailed in Table 1. As can be seen S. aureus and CNS isolates showed the highest in vitro resistance rate to penicillin G. Only 35 (10 S. aureus and 25 CNS) of the isolated strains were susceptible to all antibiotics tested, while the remaining 204 isolates were resistant at least to one of the antibiotics. Among 239 staphylococci isolated from bovine mastitis, 18 isolates of S. aureus and 31 isolates of CNS were found phenotypically resistant to methicillin. The antibiotic resistance rates of 49 MRS are shown in Table 2. Of the S. aureus isolates which showed methicillin resistance (MRce), 100% were also resistant to penicillin G, ampicillin, amoxycillin, cloxacillin, and gentamicin. MR-CNS isolates were also 100% resistant to penicillin G, ampicillin, amoxycillin and 96.8% resistant to cloxacillin. Out of 68 S. aureus isolates, 38 (55.9%) produced β-lactamase. Their resistance rates were shown in Table 3. All β-lactamase-producing isolates were susceptible to amoxycillin/clavulanic acid.
Table 1 Antibiotic susceptibility of S. aureus and coagulase negative staphylococci strains isolated from bovine mastitis CNS (n=136 isolates)
S. aureus (n=103 isolates) Resistant
Total (n=239 isolates)
Susceptible
Resistant
Susceptible
Susceptible
Resistant
n
%
n
%
n
%
n
%
n
%
n
%
Penicillin G
51
37.5
85
62.5
39
37.9
64
62.1
90
37.7
149
62.3
Ampicillin
60
44.1
76
55.9
45
43.7
58
56.3
105
43.9
134
56.1
Amoxycillin
72
52.9
64
47.1
56
54.4
47
45.6
128
53.6
111
46.4
Ampicillin/sulbactam
107
78.7
29
21.3
89
86.4
14
13.6
196
82.0
43
18.0
Amoxycillin/clavulan
132
97.1
4
2.9
96
93.2
7
6.8
228
95.4
11
4.6
Methicillin
105
77.2
31
22.8
85
82.5
18
17.5
190
79.5
49
20.5
Cloxacillin
106
77.9
30
22.1
85
82.5
18
17.5
191
79.9
48
20.1
Cefuroxime
125
91.9
11
8.1
93
90.3
10
9.7
218
91.2
21
8.8
Neomycin
94
69.1
42
30.9
92
89.3
11
10.7
186
77.8
53
22.2
Lincomycin
115
84.6
21
15.4
80
77.7
23
22.3
195
81.6
44
18.4
Enrofloxacin
132
97.1
4
2.9
99
96.1
4
3.9
231
96.7
8
3.3
Danofloxacin
132
97.1
4
2.9
98
95.1
5
4.9
230
96.2
9
3.8
Gentamicin
92
67.6
44
32.4
45
43.7
58
56.3
137
57.3
102
42.7
Trimethoprim/sulpha
85
62.5
51
37.5
56
54.4
47
45.6
141
59.0
98
41.0
93
68.4
43
31.6
40
38.8
63
61.2
133
55.6
106
44.4
Antibiotic
ic acid
methoxazole Oxytetracycline
43 Table 2 Antibiotic resistance of 49 strains of phenotypical methicillin resistant staphylococci isolated from bovine mastitis MRSA (n=18 isolates)
MR-CNS (n=31 isolates)
Antibiotic
n
%
n
%
Penicillin G
18
100.0
31
100.0
Ampicillin
18
100.0
31
100.0
Amoxycillin
18
100.0
31
100.0
Ampicillin/sulbactam
7
38.9
8
25.8
Amoxycillin/clavulanic acid
7
38.9
4
12.9
Cloxacillin
18
100.0
30
96.8
Cefuroxime
10
55.6
9
29.0
Neomycin
8
44.4
12
38.7
Lincomycin
7
38.9
10
32.3
Enrofloxacin
4
22.2
1
3.2
Danofloxacin
4
22.2
1
3.2
Gentamicin
18
100.0
14
45.2
Trimethoprim/sulphamethoxazole
11
61.1
12
38.7
Oxytetracycline
15
83.3
13
41.9
Table 3 Antibiotic resistance rates of β-lactamase positive and negative strains of S. aureus isolated from bovine mastitis
Antibiotic
\
β-lactamase positive S. aureus
β-lactamase negative S. aureus
(n = 38)
(n = 30)
Susceptible
Resistant
Susceptible
Resistant
n
%
n
%
n
%
n
%
Penicillin G
9
23.7
29
76.3
21
70.0
9
30.0
Ampicillin
10
26.3
28
73.7
24
80.0
6
20.0
Amoxycillin
19
50.0
19
50.0
25
83.3
5
16.7
Ampicillin/sulbactam
37
97.4
1
2.6
30
100.0
0
0
Amoxycillin/clavulanic acid
38
100.0
0
0
30
100.0
0
0
Methicillin
30
78.9
8
21.1
27
90.0
3
10.0
Cloxacillin
31
81.6
7
18.4
27
90.0
3
10.0
Cefuroxime
35
92.1
3
7.9
30
100.0
0
0
Neomycin
35
92.1
3
7.9
27
90.0
3
10.0
Lincomycin
30
78.9
8
21.1
21
70.0
9
30.0
Enrofloxacin
37
97.4
1
2.6
30
100.0
0
0
Danofloxacin
37
97.4
1
2.6
30
100.0
0
0
Gentamicin
16
42.1
22
57.9
13
43.3
17
56.7
Trimethoprim/sulphamethoxazole
20
52.6
18
47.4
12
40.0
18
60.0
Oxytetracycline
13
34.2
25
65.8
9
30.0
21
70.0
44
Discussion In this paper, the information on the in vitro activity of 15 antibacterial agents against staphylococcal strains isolated from bovine mastitis is presented. Susceptibility rates of staphylococcal isolates were found to be significantly lower than those reported in some countries (5, 7, 11, 18). This might be attributed to misuse of antibacterial agents in Turkey, where these are practically dispensed without a prescription. In cases of mastitis the wrong or incomplete treatment of animals also contributes significantly to the development of bacterial resistance against them. A large number of the isolates were found to be resistant to long ago established antibiotics (penicillin G, ampicillin, amoxycillin, gentamicin, oxytetracycline, trimethoprim/ sulphamethoxazole) compared to susceptibility of all isolates to the more recently developed compounds (amoxycillin/clavulanic acid, enrofloxacin, danofloxacin, cefuroxime). This result is similar to that reported in other studies for same antibiotics (14, 18). Among the 15 antibiotics tested against the isolated staphylococci, penicillin G was the least effective as similar to the results of other studies (3, 4, 6, 14). The resistance to penicillin G observed in this study must be of concern since this antibiotic represents the main antibiotic group recommended for staphylococcal mastitis treatment. Antibiotic resistance is carried on plasmids and transposons which can pass from one staphylococcal species to another (23). Regular use of antibiotics for the treatment of cows may result in the spread of resistant strains. Besides β-lactam antibiotics, gentamicin, oxytetracycline and trimethoprim/sulphamethoxazole were widely used in cows for the treatment and prevention of diseases; therefore, a high rate of resistance to these antibiotics was not unexpected. It has been reported that the most widespread mechanism of resistance to the aminoglycoside antibiotics is the modification of the antibiotic by aminoglycoside-modifying enzymes, and that these enzymes are very common in gentamicin and neomycin resistant staphylococci isolated from cases of mastitis, similarly to the strains isolated from humans (8). Although no resistance to neomycin (5) and to gentamicin (7, 11) was detected in staphylococci from bovine mastitis, we found that CNS and S. aureus isolates were more resistant to neomycin and gentamicin contrarily to those results. This contrary result may be explained by the intensive use of gentamicin and neomycin in Turkey, and by possible differences among the staphylococci isolated. MRce has been detected in S. aureus (13) and CNS (7, 18, 21) from mastitis cases in several countries. MRce of Staphylococcus strains reported here (20.5%) was higher than that found in previous studies (7, 18, 21). Furthermore, MRce seems to be more widespread among CNS than among S. aureus. This finding is supported by de Neeling et al. (2), Werckenthin et al. (23) and van Duijkeren et al. (21), who reported that MRce is more prevalent among CNS. In farms with staphylococcal mastitis problem cloxacillin is used as a dry cow treatment and its widespread use could promote the selection of resistant clones. Therefore, resistance genes could arrived into bacteria that cause infections in cows by the manipulators of the milking machines, being favoured in their dissemination by selective pressure of the antibiotics. On the other hand, it has been
reported that disk diffusion method does not identify truly methicillin-resistant (MR) isolates, and MRS should be tested for the presence of mecA gene (10). The isolates assumed to be MR by the result of the disk diffusion test were not examined by PCR for the presence of mecA gene. In this respect, we did not regard them as real MRS. MRce in staphylococci is mediated by the mecA gene, encoding the penicillin-binding protein 2a (PBP2a), which has a reduced affinity to the penicillinase-resistant penicillins such as methicillin, cloxacillin and oxacillin and for all other βlactam antibiotics (9, 10). In the present study, all the CNS and S. aureus isolates, assumed to be MR, were found resistant to β-lactam antibiotics and cloxacillin, similarly to the results of other studies (2, 7, 17, 21). Phenotypic MRce may appear in staphylococci which harbour no mecA, because they produce large amounts of β-lactamase. These types of resistance usually appear as low-level or border-line resistance and addition of β-lactamase inhibitors such as sulbactam or clavulanate may help overcome MRce (9, 10). In the study, out of 68 S. aureus isolates studied were 55.9% β-lactamase producer. Penicillin and ampicillin were most affected by β-lactamase activity, similarly the other reported studies (20, 22). β-lactamase producer isolates were 100% susceptible to amoxycillin/clavulanic acid and 97.4% susceptible to ampicillin/sulbactam, but were 21.1% resistant to methicillin. We considered that the amount of β-lactamase produced may cause this result as described by Craven et al. (1). It has been reported that most cephalosporins and penicillinase-resistant penicillins were effective against βlactamase-producing staphylococci (1, 4, 22). In this study, the susceptibility rates to cefuroxime, cloxacillin, and methicillin of 38 β-lactamase-producing S. aureus were 91.2%, 81.6%, and 78.9%, respectively. In addition, it was observed that the susceptibility rate to methicillin of the βlactamase-producing S. aureus isolates was lower than to cefuroxime and cloxacillin. This is reasonable, because methicillin disks are not well storable under normal storage condition (16). In conclusion, the differences observed in the activity of the antibiotics tested against staphylococci show the importance of antibiotic susceptibility tests, performed together with the identification of the bacterial agents. In the treatment of the infected animals, it is important to determine the phenotype of resistance and to avoid the selection of resistant strains.
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